The development and survival of neurons depends upon an intricate network of interactions between cells. The differentiation of each of the hundreds of cell types in the nervous system results from the expression of a specific set of genes in response to multiple extracellular and intracellular signals. One of the few neuronal determination genes known in the mammalian nervous system is that encoding the transcription factor MASH 1. MASH 1 is expressed transiently in subsets of early precursor cells in both the central and peripheral nervous systems and is essential in the development of particular neurons. Transgenic mice lacking the normal gene are missing neurons in the sympathetic, parasympathetic, enteric, and olfactory nervous systems. To understand the molecular mechanism of neuronal development, we must learn what regulates the expression of this gene. The first goal of the research proposed here is to identify the cis-acting elements that regulate the transcription of MASH1. This will be accomplished by assaying the ability of DNA sequences 5' and 3' to the protein-coding region of the MASH 1 gene to direct the expression of a reporter gene in a spatial and temporal pattern identical to that for MASH 1 expression in cell culture and transgenic mice. Because retinoic acid can induce MASH 1 expression, the cis-acting regulatory elements responsive to this inducer will also be defined. Once the regulatory elements in the MASH 1 locus have been delineated, they will be used as probes to identify the proteins that bind them and that regulate the pattern of MASH 1 expression. We shall finally screen cDNA expression libraries to identify the genes encoding such DNA-binding proteins. By defining the factors that mediate the earliest stages of neural specification, identification of these proteins will provide a better understanding of the molecular mechanisms important in development of the mammalian nervous system.